1. Field of the Invention
This invention relates to a method and reagents for detecting amphetamine-class drugs in a test sample such as urine. In particular, the invention relates to a fluorescence polarization immunoassay procedure for determining the presence or amount of amphetamine-class drugs in a test sample, to a novel class of tracer compounds used as reagents in such procedures, and to immunogen compounds used to raise antibodies for use in such procedures.
2. Description of Related Art
Amphetamine-class drugs are sympathomimetic phenethylamine derivatives having central nervous system stimulant activity. These drugs have been used for the treatment of obesity, narcolepsy and hypotension. Excessive use of these drugs, however, may lead to tolerance and physical dependence, and because of their stimulant effects the drugs are commonly abused. Physiological symptoms often associated with very high amounts of ingested amphetamine-class drugs include elevated blood pressure, dilated pupils, hyperthermia, convulsions and acute amphetamine psychosis.
The biological fluid used most frequently for detecting or quantitating amphetamine-class drugs is urine. Other biological fluids, however, such as serum, plasma or saliva might be used as test samples. In the past, amphetamines have been detected by a number of techniques including thin-layer chromatography (TLC), gas chromatography (GC) and high performance liquid chromatography (HPLC). These methods generally involve complicated chemical extractions of the drugs from the test sample, procedures which require trained personnel and lengthy assay times.
Binding assays are a preferred alternative to the chemical methods such as GC, TLC and HPLC for the detection of analytes. Binding assays for detecting antigens and antibodies depend upon the immunological reactivity which characterizes these substances. Generally, these assays are collectively termed immunoassays.
Immunoassay techniques take advantage of the mechanisms of the immune systems of higher organisms, wherein antibodies are produced in response to the presence of antigens which are pathogenic or foreign to the organisms. One or more antibodies are produced in response to and are reactive with a particular antigen, thereby creating a highly specific reaction mechanism which can be used in vitro to determine the presence or concentration of that particular antigen in a biological sample.
Competitive binding immunoassays for measuring analytes of interest are based on the competition between the analyte in the test sample and a labeled reagent (i.e., tracer) for a limited number of binding sites on a binding member (e.g., an antibody) that is specific for both the analyte and tracer. Generally, the concentration of analyte in the sample determines the amount of tracer that will bind to the antibody. The amount of tracer/antibody complex produced can be quantitatively measured and is inversely proportional to the quantity of analyte in the test sample.
Fluorescence polarization provides a means for measuring the amount of tracer/antibody complex produced in a competitive binding immunoassay. Fluorescence polarization techniques are based on the principle that, when excited by linearly polarized light, a fluorescently labeled reagent will rotate rapidly, and fluorescent light emitted by that rotating tracer becomes partially depolarized due to the rapid rotation. As a result, the tracer will emit fluorescence with a degree of polarization inversely related to the tracer's rate of rotation, i.e., the higher the rotation the lower the polarization of the emitted light (or the greater the depolarization of the emitted light). The speed of rotation and the amount of depolarization decrease when the tracer becomes bound to a heavier molecule, such as when it becomes bound to the comparatively heavier antibody molecule. If a reaction mixture containing a fluorescent tracer/antibody complex is excited by linearly polarized light, then the emitted light generally remains polarized because the fluorophore in the complex is constrained from rapidly rotating. When a "free" tracer (i.e., tracer that is not bound to an antibody) is excited by linearly polarized light, its rotation is much faster than that of the tracer/antibody complex, and therefore, depolarization of the emitted light is increased.
By comparing standard preparations containing known concentrations of analyte to test samples containing unknown levels of the analyte, the fluorescence polarization technique provides a quantitative means for measuring the amount of tracer/antibody complex produced in a competitive binding assay. This technique is currently being employed by Abbott Laboratories in its commercially available TDx.RTM. Therapeutic Drug Monitoring System (as described in U.S. Pat. No. 4,269,511 and U.S. Pat. No. 4,420,568) and its commercially available IMx.RTM. and ADx.TM. automated instruments.
As disclosed in the '511 and '568 patents, because the tracer must compete with the analyte for binding to the antibody in a fluorescence polarization immunoassay (FPIA), the tracer must possess a molecular structure sufficiently similar to the analyte so as to be recognized by an antibody specific for the analyte. For this reason the tracer is also referred to as a fluorescently labeled analyte-analog, a substantial portion of which has the same spatial and polar organization as the analyte to define one or more determinant sites capable of competing with the analyte for the binding sites on the antibody.
An accurate and reliable immunoassay for the detection or quantification of a specific amphetamine-class compound requires that antibody cross-reactivity, i.e., the recognition of compounds other than the desired analyte, be minimized. Copending U.S. Patent Application Ser. Nos. 010,355 (filed Feb. 3, 1985) and 265,361 (filed Oct. 28, 1988) disclose assay reagents and FPIA methods for quantitating amphetamine and methamphetamine in test samples while eliminating phenethylamine cross-reactivity.
To date, however, no fluorescence polarization immunoassay has been disclosed which enables the screening of a test sample for a broad range of amphetamine-class drugs. Accordingly, a need exists for providing an assay and reagents for performing an accurate and sensitive FPIA for the simultaneous detection of the presence or amount of amphetamine-class drugs.